Orthoester protecting groups

ABSTRACT

Novel orthoesters are provided which can be used as a 2&#39;-hydroxyl protecting groups or 2&#39;-modification in the synthesis of polymers containing ribonucleic acid (RNA) nucleotides. The RNA comprising the orthoester can be handled and analyzed while 2&#39;-modified, thereby minimizing potential degradation. The orthoester is stable during oligonucleotide synthesis. The orthoester is subsequently modified and can then be removed under mild acidic conditions. The ease and dependability of this process and the quality of the RNA product synthesized with this invention are comparable to that previously associated only with DNA synthesis.

TECHNICAL FIELD

The present invention relates to the field of protecting groups inorganic synthesis and, more particularly, to the use of these compoundsas ribonucleoside protecting groups and as 2'-modifications. Still morespecifically, the protecting groups are used in the synthesis ofoligonucleotides containing ribonucleotide subunits.

BACKGROUND OF THE INVENTION

The ability to routinely synthesize ribonucleic acid (RNA) has becomeincreasingly important as research reveals the multitude of RNA'sbiological functions. There are many types of RNA including ribosomalRNA, transfer RNA and messenger RNA. RNA also is important in variousstructures and functions as well as being a catalyst in enzymaticreactions, as in the case of ribozymes. Because of the importantbiological roles RNA plays, both known and unknown, it is ofconsiderable utility to be able to synthesize short (2-300 nucleotides)defined sequences of RNA, commonly referred to as RNA oligonucleotidesor oligoribonucleotides. Over the past 25 years, many chemicalapproaches have been explored for synthesizing RNA oligonucleotides.Because deoxyribonucleic acid (DNA) methodologies have progressed morerapidly, the usual strategy for the synthesis of RNA has been to adaptDNA chemistries to RNA synthesis. Consequently, most approaches havefocused on retaining the 5'-dimethoxytrityl (DMT) ether and adding acompatible 2'-hydroxyl protecting group such as fluoride-labile silylethers, photo-labile moieties, and acid-labile acetals. A delicatebalance has been required to successfully utilize the acid-labile2'-acetals in conjunction with the acid-labile 5'-DMT ether. Therefore,other approaches have involved retaining the 2'-acetal while replacingthe 5'-DMT.

The acid-labile acetals have many attractive features. For example, ithas been reported that it is possible to chromatograph some2'-acetal-protected RNA. However, the subsequent removal of theseacetals, which are used with the DMT group, requires acidic conditionswhich subsequently cause degradation of the RNA, or require extremelylong periods of time to remove (>24 hours). Therefore, although it maybe possible to safely handle and purify some 2'-acetal-protected RNAs,the harsh conditions required for rapid deprotection may require furtherpurification of the RNA, thereby negating this advantage. Milderconditions can be used but these are inconvenient, requiring more than24 hours. More-labile acetals can not be used as they would not besufficiently stable to the DMT acid-deprotection conditions during RNAsynthesis.

Of all of the RNA synthesis methods reported to date, only the5'-DMT-2'-t-butyldimethylsilyl (TBDMS) and the5'-DMT-2'-[1-(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP)chemistries are readily available commercially. Unfortunately, neitherof these methods allows RNA synthesis to be as routine and dependable asDNA synthesis. The impediments facing 5'-DMT-2'-FPMP chemistry arerelated to the problem of balancing two acid-labile protecting groups.One of the major difficulties with the 2'-TBDMS approach is thatstepwise coupling yields are only 96-98% under routine conditionscompared to >99% for DNA. These methods enable the synthesis of RNA inacceptable yields and quality, but a high level of skill and significantinvestments in training and experience are required to deliver adequateresults.

One of the most desirable conditions for the final 2'-deprotection ofsynthesized RNA is an extremely-mildly-acidic aqueous solution. In theoptimal scheme, the 2'-protecting groups only have to be stable towithstand oligonucleotide synthesis conditions. Scaringe and Caruthers(U.S. patent application Ser. No. 08/488,878, filed Jun. 9, 1995 andincorporated herein by reference) recently reported a novel RNAsynthesis strategy similar to such a scheme. Their investigations led tothe development of silyl ethers for protection of the 5'-hydroxyl.However, this 5'-silyl ether oligonucleotide synthesis chemistry was notcompatible with mildly-acid-labile 2'-acetals. Acid-labile orthoesterprotecting groups were investigated and discovered to have potential foruse at the 2'-hydroxyl. The 2'-orthoesters that were developed inconjunction with 5'-silyl ethers enabled the synthesis of RNAoligonucleotides. Scaringe and Caruthers disclose specific orthoesterprotecting groups at the 2'-position of ribonucleotides. However, theydo not disclose the orthoesters of the present invention.

The present invention provides an orthoester moiety that serves as aprotecting group, particularly for RNA synthesis. Also provided in thisinvention is RNA comprising the protecting group which possesses noveladvantages and useful features, e.g., a modified RNA oligonucleotidethat is easily handled and analyzed with minimal concern aboutdegradation. The protecting groups can be readily cleaved (<10 minutes),if so desired, under extremely mild conditions that cause no detectabledegradation of the RNA. No prior art anticipates that the following2'-modification ##STR1## would be advantageous to RNA synthesis. This2'-modification is the result of using an orthoester of the followinggeneral structure: ##STR2## where R represents protecting groups whichcan be removed prior to removing the orthoester.

The prior art has provided several means to synthesize RNAoligonucleotides. However, none have enabled the synthesis, handling,analysis and use of RNA oligonucleotides to be as robust and dependableas DNA synthesis, nor produced RNA comparable to the high quality inwhich DNA can be produced. No prior art has disclosed a modification ofRNA that enables the RNA to be easily handled and then where themodification, e.g., a protecting group, can be removed under mildconditions to yield fully deprotected RNA. The present invention,therefore, provides more robust RNA synthesis methods which consistentlyproduce higher quality RNA on a routine basis.

SUMMARY OF THE INVENTION

Accordingly, it is an object of this invention to provide usefulprotecting groups for the improved synthesis, analysis, handling and useof RNA oligonucleotides or other polymers containing ribonucleotides. Itis a further objective of this invention to provide RNA, comprising suchprotecting groups, that can be easily analyzed, handled and used withoutrequiring extensive safeguards against degradation. The protectinggroups can be subsequently removed if desired under extremely mildconditions to yield high-quality fully deprotected RNA. Those skilled inthe art can use these protecting groups in other organic synthesismethodologies as well. Therefore, this invention is not limited to thefield of nucleic acid and oligonucleotide chemistry.

The present invention achieves these and other objectives by theprovision of novel orthoester protecting groups with innovative anduseful features. We have developed, for example, theO-bis(2-acetyl-ethoxy)methyl (ACE) orthoester: ##STR3## that is stableto nucleoside and oligonucleotide synthesis conditions but is modifiedvia ester hydrolysis during base deprotection of the oligonucleotide orpolymer. The resulting 2'-bis(2-hydroxyethyl)methyl orthoesterprotecting group (2'-EG): ##STR4## is 10 times more labile to acid thanit's precursor, the ACE orthoester. Complete cleavage of the 2'-EGorthoester may be effected using extremely mild conditions (pH 3, <10min., 55° C.). The innovative features of this chemistry have enabledthe synthesis of RNA oligonucleotides surpassing results in the priorart in terms of quality.

The orthoesters of the present invention are illustrated as follows:##STR5## where R represents protecting groups which can be removed priorto removing the orthoester. An example of an orthoester with appropriateR groups is as follows: ##STR6## where X₁,X₂, and X₃ are appropriateatoms or ligands. Several suitable orthoesters are illustrated asfollows: ##STR7##

The general structure of a ribonucleoside of this invention isillustrated as follows: ##STR8## where exocyclic amines, 5'-hydroxyl andthe 3'-hydroxyl groups are appropriately protected and/or functionalizedfor use in oligonucleotide synthesis and R represents protecting groupswhich can be removed while leaving the 2'-orthoester moiety intact. Morespecifically, the general structure of a ribonucleoside of thisinvention has the following structure where the R groups from above areacyl protecting groups and X₁,X₂, and X₃ are appropriate atoms orligands: ##STR9##

The ACE orthoester comprises protecting groups. No prior art describesthe use of protected orthoesters in oligonucleotide synthesis. It wasnot known that an orthoester utilizing, for example, protected ethyleneglycol ligands would be stable to oligonucleotide synthesis conditions.It also was not known that orthoesters would be significantly morelabile once protecting groups were removed from the ethylene glycolligands. The novel orthoesters have been utilized for the synthesis ofRNA of higher quality than that disclosed in prior art. Furthermore,this invention has made it possible to synthesize, handle, analyze anduse RNA with ease comparable to DNA. Some useful aspects of thisinvention are as follows:

(1) It is possible to analyze and handle RNA while it is still2'-protected. The structure of a typical ribonucleotide in a polymer ofthe present invention has the 2'-EG modified structure: ##STR10## Theability to analyze and handle polymers containing the 2'-EG-modifiedribonucleotide subunit is important for two major reasons: (a) The2'-modified RNA is relatively stable to degradation. Therefore, it isnot necessary to observe stringent sterile conditions while handling,analyzing and purifying 2'-modified RNA synthesized using thisinvention. (b) While the RNA is still 2'-modified, it is possible toanalyze and purify the oligonucleotides. Over 200 sequences have beensynthesized according to this invention and it has been possible toresolve every oligonucleotide into a major product during analysis. (Tothose skilled in the art, it is known that a percentage of RNAoligonucleotides, approximately 5-10%, can not be resolved under routineanalysis conditions if the RNA is fully deprotected because of strongsecondary structures and folding common to RNA.) Thus, it is possible tosynthesize RNA sequences without concern over whether it will bepossible to analyze the final product.

(2) The RNA quality is consistent with any sequence. For comparison, ithas been reported that a 27-mer was synthesized with standard5'-silyl-2'-orthoester chemistry in 35% overall yield. The same 27-merwas synthesized with 5'-DMT-2'-TBDMS chemistry in 45% overall yield.Using a novel orthoester of the invention, a comparable 27-mer wassynthesized in >70% overall yield. A 36-mer described in a followingexample was routinely synthesized in 65-70% overall yield. Couplingyields of >99% were possible in <90seconds. For many applications usingRNA synthesized with this invention, it is no longer necessary to purifythe RNA after synthesis as the quality of the crude RNA synthesized issufficient for subsequent use. The high quality and high yields of RNAobserved with such short coupling times are comparable with thoseroutinely experienced in DNA synthesis.

(3) The final 2'-deprotection conditions of the RNA oligonucleotide arethe mildest ever reported for this application by an order of magnitude.For example, 2'-ACE-uridine has a half life of ˜7.5 minutes at pH 2, 25°C., which is comparable to previously reported orthoesters and acetalsthat have been used in oligonucleotide synthesis. However, when the ACEorthoester is modified by ester hydrolysis, the 2'-modified uridine is10 times more labile with a half life of <45 seconds at pH 2, 25° C.Several assays demonstrated that there was no detectable degradation orisomerization under the extremely mild conditions used to 2'-deprotectRNA oligonucleotides synthesized according to this invention.

This invention includes several other protecting groups. For example,the following 2'-acetal protecting group is included within thisinvention and can be synthesized by those skilled in the art utilizingthe present specification and well-known synthetic techniques. ##STR11##This protecting group, or variations thereof, would exhibit similarproperties to the orthoester protecting groups of this invention.Therefore, this invention includes other classes of protecting groups.The present invention also provides polymers and oligonucleotides thatcomprise an acid-labile protecting group wherein the half life of theprotecting group is as follows: the half life of that protecting group,when on the 2'-hydroxyl of a uridine nucleoside, is <3 minutes at pH 2,25° C.

The invention also provides oligonucleotide comprising an acid-labile2'-hydroxyl protecting group wherein the half life of the protectinggroup is as follows: the half life of that protecting group, when on the2'-hydroxyl of a uridine nucleoside, is <3 minutes at pH 2, 25° C.

Further, the invention provides an oligonucleotide comprising anacid-labile 2'-hydroxyl protecting group which is itself protected by asecond protecting group, and, wherein upon removal of the secondprotecting group(s) on the first protecting group, yield an acid-labilefirst protecting group wherein the half life of the first protectinggroup is now as follows: the half life of the deprotected firstprotecting group, when on the 2'-hydroxyl of a uridine nucleoside, is <3minutes at pH 2, 25° C.

The features of the novel orthoesters of the present invention have madeit possible to routinely synthesize RNA in high quality. Followingsynthesis, it is now possible to analyze almost any RNA oligonucleotide.Prior to this invention, this was not always possible. The RNAsynthesized with this invention can be handled without the need forsterile conditions. When ready for use, the RNA is easily deprotectedunder extremely mild conditions that do not degrade the RNA norcontribute any detectable impurities. Oligonucleotides and polymerssynthesized with this invention may be used for a wide array of purposesin various applications, including as antisense molecules, enzymaticmolecules, diagnostic molecules, therapeutic molecules and researchmolecules. All of these uses are well known to those skilled in the art.The 2'-modification can be left on for subsequent applications, forexample, analysis and purification, or it may be removed, if desired, toyield RNA with 2'-hydroxyl groups.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Anion-exchange HPLC chromatograph of unpurified 2'-protected SEQID NO: 1 (2'-ACE chemistry)

FIG. 2. Anion-exchange HPLC chromatograph of fully deprotected SEQ IDNO: 1 (2'-ACE chemistry)

FIG. 3. Anion-exchange HPLC chromatograph of fully deprotected SEQ IDNO: 1 (5'-DMT-2'-TBDMS chemistry)

FIG. 4. Anion-exchange HPLC chromatograph of unpurified 2'-protected SEQID NO: 2

FIG. 5. Anion-exchange HPLC chromatograph of fully deprotected SEQ IDNO: 2

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following terms have the specified meanings:

By "atom" is meant a single element unit, either neutral or with charge,that is appropriate for the indicated position within the structure. Forexample, an "atom" with one bond to it may be chlorine, or hydrogen, oroxygen with a single minus charge. An "atom" with two bonds to it can beoxygen, or sulfur, or nitrogen with a minus charge.

By "ligand" is meant an organic structure comprising up to 30 atoms (notincluding hydrogen) of which the majority are carbon, oxygen, nitrogen.

By "orthoester" is meant a moiety or molecule comprising the followinggeneric structure: ##STR12## i.e., wherein three oxygen atoms are bondedto a central carbon atom and R is an atom or ligand, preferablyhydrogen.

By "oligonucleotide" is meant a molecule comprising two or morenucleotides. The polynucleotide can be single, double or multiplestranded and may comprise modified or unmodified nucleotides ornon-nucleotides or various mixtures and combinations thereof. Preferablythe oligonucleotide comprises about 2-50 nucleotides.

By "polymer" is meant a molecule containing one or more types ofsubunits which may occur one, two or multiple times within the molecule,e.g. a ribonucleic acid, a peptide-nucleic acid hybrid.

By "phosphorus moiety" is meant a group of atoms comprising one or morephosphorus atoms. Preferably, the total number of all atoms in thisgroup is less than 40 (not including hydrogen).

By "protecting group" is meant a group of atoms which purpose is totemporarily mask the functionality of the site to which it is attachedon a molecule. Prior to the use of the molecule in a subsequent analysisor application, the protecting group may or may not be removed.

By "functional group" is meant a site on a molecule that has, as knownto those skilled in the art, the potential to participate in reactions.Functional groups include, for example, hydroxyls, amines, thiols,halogens, phosphoramidites.

By "nucleotide" is meant, as recognized in the art, natural bases(standard), and modified bases well known in the art. Such bases aregenerally located at the 1' position of a sugar moiety. A nucleotidegenerally comprises a base, sugar and a phosphate group. The nucleotidescan be unmodified or modified at the sugar, phosphate and/or base moiety(also referred to interchangeably as nucleotide analogs, modifiednucleotides, non-natural nucleotides, non-standard nucleotides andother).

The symbol as used in structural drawings represents that the line thatis immediately perpendicular to this symbol is a bond to another atom oratom within a larger molecule.

By "phosphoramidite" is meant the functional moiety as first disclosedby Caruthers and Beaucage (U.S. Pat. No. 4,415,732).

By "enzymatic nucleic acid molecule" is meant a molecule, comprising atleast one nucleic acid, capable of catalyzing (altering the velocityand/or rate of) one or more reactions, for example, the cleavage ofseparate nucleic acid molecules in a nucleotide specific manner. Theterm enzymatic nucleic acid molecule can be used interchangeably, forexample, with phrases such as ribozymes, catalytic RNA, enzymatic RNA,catalytic DNA, catalytic oligonucleotides, nucleozyme, DNAzyme, RNAenzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme,or DNA enzyme.

By "modified nucleoside" or "modified nucleotide" is meant anynucleoside or nucleotide subunit which contains a modification in thechemical structure of the unmodified base, sugar and/or phosphate.

The general process for utilizing this invention is as follows.Nucleosides are suitably protected and functionalized for use insolid-phase or solution-phase synthesis of RNA oligonucleotides. The2'-hydroxyl group in a ribonucleotide is modified using a trisorthoester reagent of this invention. (The 2'-hydroxyl is modified toyield a 2'-O-orthoester nucleoside of this invention by reacting theribonucleoside with the tris orthoester reagent in the presence of anacidic catalyst, e.g., pyridinium p-toluene sulfonate. This reaction isknown to those skilled in the art.) The product is then subjected tofurther protecting group reactions (e.g., 5'-O-silylation) andfunctionalizations (e.g., 3'-O-phosphitylation) to produce a desiredreagent (e.g., nucleoside phosphoramidite) for incorporation within anoligonucleotide or polymer by reactions known to those skilled in theart.

A preferred embodiment of this invention is an orthoester comprisingethylene glycol ligands which are protected with acyl or esterprotecting groups. More specifically, the preferred acyl group isacetyl. The nucleoside reagents may then be used by those skilled in theart to synthesize RNA oligonucleotides on commercially availablesynthesizer instruments, e.g. Gene Assembler Plus (Pharmacia), 380B(Applied Biosystems). Following synthesis (either solution-phase orsolid-phase) of an oligonucleotide or polymer using a compound of thisinvention, the product is subjected to one or more reactions usingnon-acidic reagents. One of these reactions may be strong basicconditions, for example, 40% methylamine in water for 10 minutes at 55°C., which will remove the acyl protecting groups from the ethyleneglycol ligands but leave the orthoester moiety attached. The resultantorthoester may be left attached when the polymer or oligonucleotide isused in subsequent applications, or it may be removed in a finalmildly-acidic reaction, for example, 10 minutes at 55° C. in 50 mMacetic acid, pH 3.0, followed by addition of equal volume of 150 mM TRISbuffer for 10 minutes at 55° C.

The following examples are meant to be exemplary only and not limitingin any way.

EXAMPLE I Synthesis of orthoester reagent and5'-silyl-2'-ACE-3'-O-(N,N-diisopropylamine)-methoxyphosphine-uridine.

The reagents in this example can be obtained from a variety ofcommercial sources, e.g., Aldrich Chemical (Milwaukee, Wis.), TCIAmerica (Portland, Oreg.) and Monomer Sciences (New Market, Ala.).

Synthesis of tris(2-acetyl-ethoxy) orthoformate, ACE orthoester reagent:Acetic acid ethyl ester (85%) (5 eq., 323 g) was treated with pyridiniump-toluene sulfonate (0.2 eq, 30.8 g) and trimethyl orthoformate (1 eq.,67.8 ml). The reaction was heated to distill off the methanol product.The reaction was cooled and then neutralized with base. The product waspurified by column chromatography and high vacuum distillation. Finalyield of product was 20%.

Synthesis of 2'-O-bis(2-acetyl-ethoxy)methyl uridine (representative ofgeneral 2'-protection reaction): 5'-O-3'-O-tetraisopropyldisiloxyluridine (TIPS-uridine) (1 eq., 4.86 g) was reacted neat withtris(2-acetyl-ethoxy) orthoformate (2.8 eq., 9 g) and pyridiniump-toluene sulfonate (0.2 eq., 0.5 g) at 55° C. for 3 hours under highvacuum (<15 microns of Hg). The reaction was cooled to room temperatureand neutralized with base. The crude reaction was passed over silica gelto do a crude purification to remove the neutralized catalyst. Theenriched mixture was treated with a premixed solution ofN,N,N',N'-tetramethylethylendiamine (TEMED) (9.05 ml) and 48%hydrofluoric acid (1.08 ml) in acetonitrile (100 ml) for 6 hours. Theproduct, 2'-O-bis(2-acetyl-ethoxy)methyl uridine, was purified by columnchromatography. The yield for the combined two reactions was 65%.Adenosine (N-benzoyl), cytidine (N-acetyl) and guanosine (N-isobutyrl)2'-ACE nucleosides were similarly synthesized and similarly carriedthrough the next two reactions to produce final nucleosidephosphoramidites for use in RNA synthesis.

Synthesis of 5'-O-silyl-2'-O-ACE-uridine: To 2'-O-ACE-uridine (1 eq.,4.9 g) and imidazole (4 eq., 2.8 g) in tetrahydrofuran was addedbis(trimethylsiloxy)-cyclooctoxy-silylchloride (OCT-C1) (1.5 eq., 5.86 gin 20 ml tetrahydrofuran) over 30 minutes with stirring. OCT-C1 can besynthesized by those skilled in the art frombis(trimethylsiloxy)-dichlorosilane and cyclooctanol. The5'-silyl-2'-ACE uridine product was purified by silica gelchromatography and isolated in 75-85% yield.

Synthesis of 5'-O-silyl-2'-ACE-uridine-3'-O-(N,N-diisopropylmethoxy)phosphoramidite:To a solution of 5'-O-silyl-2'-O-ACE-uridine (1 eq., 6 g) in 20 ml ofdichloromethane was added firstbis(N,N-diisopropylamine)methoxy-phosphine (1.3 eq., 2.7 g) followed bytetrazole (0.8 eq., 0.45 g) with stirring. After 2 hours the reactionwas quenched and the product isolated in 80-90% yield via silica gelchromatography.

EXAMPLE II Synthesis of Oligonucleotide 36 Bases in Length (SEQ ID NO:1).

Oligonucleotide synthesis conditions were adapted from Scaringe andCaruthers, supra. Syntheses were performed on derivatized polymersupports using either a Gene Assembler Plus synthesizer (Pharmacia) or a380B synthesizer (ABI). The protocols can be adapted by those skilled inthe art to any commercially available synthesizer. The following changeswere made to the protocols of Scaringe and Caruthers. The silyldeprotection reagent was replaced by 1.0 M aqueous HF, 1.6 Mtriethylamine (TEA) in dimethylformamide (DMF). The reaction time was30-35 seconds. Oxidation was effected during every cycle using 3 Mt-butylhydroperoxide (tBuOOH) in toluene. The wash solvents, DMF andacetonitrile (MeCN), were used between the reactions. The synthesiscycle is as follows:

    ______________________________________                                        Reaction     Reagent          Time (Seconds)                                  ______________________________________                                        5'-silyl deprotection                                                                      1.0 M HF & 1.6 M TEA in                                                                        30-35                                              DMF                                                                          Wash DMF 10                                                                   Wash MeCN 40                                                                  Couple 15 eq. amidite/100 eq. S- 90                                            ethyl-tetrazole                                                              Wash MeCN 30                                                                  Oxidize tBuOOH 40                                                             Wash MeCN 30                                                                  Capping 10% acetic anhydride & 10% 30                                          N-Methyl imidazole                                                           Wash MeCN 30                                                                  Wash DMF  5                                                                 ______________________________________                                    

Following synthesis on the synthesizer, the polymer support is treatedfor 30 minutes using a 1 M solution ofdisodium-2-cobamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂ Na₂)in DMF. For a 0.2 micromole synthesis of SEQ ID NO: 1, 1 ml of thisreagent was used to cleave the methyl protecting groups from thephosphates. The S₂ Na₂ reagent was washed out with water and acetone.The dried support was treated with 1 ml of 40% N-methylamine in waterfor 10 minutes at 55° C. to cleave all base-labile protecting groups andrelease the 2'-protected SEQ ID NO: 1 RNA oligonucleotide into solution.The crude reaction mixture was analyzed by anion exchange high pressureliquid chromatography (HPLC) and the result illustrated in FIG. 1. Fromthese results it can be seen that the 2'-protected oligonucleotide canbe clearly analyzed by HPLC. The crude SEQ ID NO: 1 reaction inmethylamine and water was dried down in vacuo. The pellet wasresuspended in 1.6 ml of 50 mM acetic acid, pH 3.0, and incubated for 10minutes at 55° C. To this was added 1.6 ml of 150 mM TRIS, pH 8.7,(Final pH of solution 7.7-8.0) for 10 minutes at 55° C. An aliquot ofthis solution was then analyzed by identical HPLC conditions and theresult illustrated in FIG. 2. The same SEQ ID NO: 1 was synthesized andprovided from a commercial source which synthesized the oligonucleotideusing commercially available 5'-DMT-2'-TBDMS chemistry. The crudeproduct was analyzed under identical HPLC conditions and the resultillustrated in FIG. 3. Direct analytical comparison of the crude SEQ IDNO: 1 produced using this invention (FIG. 2) and the currentstate-of-the-art 5'-DMT-2'-TBDMS chemistry (FIG. 3) demonstrates theimproved quality now possible with this invention.

EXAMPLE III Synthesis and HPLC analysis of SEQ ID NO: 2

SEQ ID NO: 2 was synthesized using the methodology in Example II above.The 2'-orthoester crude RNA was analyzed by HPLC (FIG. 4). The HPLCresult illustrates a clear major product in 85% yield. The 2'-orthoestercrude RNA was treated as in example II to remove the 2'-orthoestermodification. The product was then analyzed under identical HPLCconditions (FIG. 5) but no major product was observed. The product wasalso analyzed under highly denaturing conditions using polyacrylamidegel electrophoresis (PAGE) with 7 M urea at 60° C. In this analysis amajor distinct product band was observed as would be expected for a14-nucleotide RNA oligonucleotide (results not shown).

All other commercially available means of synthesizing RNA produce RNAthat can only be easily analyzed when fully deprotected. When the finalRNA product can not be easily analyzed, then these results illustratethe need for this invention to provide a dependable and conclusive meansto analyze RNA oligonucleotides. As this example demonstrates it is ofsignificant utility to use this invention to be able to handle andanalyze RNA while still 2'-modified.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 2                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 36                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Other       nucleic                                                                               acid                                                                     - - <400> SEQUENCE: 1                                                         - - ucuccaucug augaggccga aaggccgaaa aucccc      - #                       - #       36                                                                     - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 14                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Other       nucleic                                                                               acid                                                                     - - <400> SEQUENCE: 2                                                         - - gggaacgucu aggg              - #                  - #                      - #     14                                                                __________________________________________________________________________

What is claimed is:
 1. An oligonucleotide comprising a moiety of theformula: ##STR13## wherein R is any appropriate ligand that protects theoxygen.
 2. An oligonucleotide comprising the structure: ##STR14##wherein X₁, X₂, X₃ are the same or different and are any atoms, selectedfrom the group consisting of Cl, H, O, S and N or appropriate ligands.3. An oligonucleotide according to claim 2 wherein X₁, X₂, X₃ arehydrogen.
 4. An oligonucleotide according to claim 1 wherein R ishydrogen.
 5. An oligonucleotide wherein a functional group has attachedthereto the structure: ##STR15## wherein R is hydrogen or anyappropriate ligand that protects the oxygen.
 6. An oligonucleotidecomprising the following structure: ##STR16## wherein R₃ is anyappropriate ligand, X₄ and X₅ are the same or different atom, selectedfrom the group consisting of Cl, H, O, S and N and wherein X₁, X₂, X₃are the same or different and are any atoms, selected from the groupconsisting of Cl, H, O, S and N or appropriate ligands.
 7. Anoligonucleotide according to claim 6 wherein X₄ and X₅ are oxygen.
 8. Anoligonucleotide according to claim 6 wherein R₃ is nothing.
 9. Anoligonucleotide according to claim 6 wherein X₄ and X₅ are oxygen. 10.An oligonucleotide according to claim 6 wherein X₄ and X₅ are oxygen andwherein X₁, X₂, and X₃ are hydrogen.
 11. An oligonucleotide according toclaim 10 wherein R₃ is nothing.
 12. An oligonucleotide comprising thefollowing structure, ##STR17## wherein X₄ and X₅ are the same ordifferent atom, selected from the group consisting of Cl, H, O, S and N.13. An oligonucleotide according to claim 12 wherein X₄ and X₅ areoxygen.
 14. A process to synthesize polymers or oligonucleotides whichutilizes any of the compounds, polymers or oligonucleotides according toany of the preceding claims.
 15. A process to synthesize polymers oroligonucleotides which utilizes any of the compounds, structures andmodifications as in any of the preceding claims and wherein theinternucleotide linkage(s) is(are) oxidized before completion ofoligonucleotide synthesis.
 16. A process according to claim 15 whereinthe internucleotide linkage is oxidized every cycle or the majority ofcycles.
 17. A compound of the formula: ##STR18## wherein R is selectedfrom the group consisting of an ester, carbonate, silyl ether andsulfonate.
 18. A compound according to claim 17 wherein R is ##STR19##wherein X₁, X₂, X₃ are the same or different and are any atoms orligands.
 19. A compound according to claim 18 wherein X₁, X₂, X₃ arehydrogen.